Yeast organisms produce a number of proteins synthesized intracellularly, but having a function outside the cell. Such extracellular proteins are referred to as secreted proteins. These secreted proteins are expressed initially inside the cell in a precursor or a pre-protein form containing a presequence ensuring effective direction of the expressed product across the membrane of the endoplasmic reticulum (ER). The presequence, normally named a signal peptide, is generally cleaved off from the desired product during translocation. Once entered in the secretory pathway, the protein is transported to the Golgi apparatus. From the Golgi the protein can follow different routes that lead to compartments such as the cell vacuole or the cell membrane, or it can be routed out of the cell to be secreted to the external medium (Pfeffer, S. R. and Rothman, J. E. Ann. Rev. Biochem. 56 (1987), 829-852).
Several approaches have been suggested for the expression and secretion in yeast of proteins heterologous to yeast. European published patent application No. 0088632A describes a process by which proteins heterologous to yeast are expressed, processed and secreted by transforming a yeast organism with an expression vehicle harbouring DNA encoding the desired protein and a signal peptide, preparing a culture of the transformed organism, growing the culture and recovering the protein from the culture medium. The signal peptide may be the desired proteins own signal peptide, a heterologous signal peptide or a hybrid of native and heterologous signal peptide.
A problem encountered with the use of signal peptides heterologous to yeast might be that the heterologous signal peptide does not ensure efficient translocation and/or cleavage after the signal peptide.
The S. cerevisiae MF.alpha.1 (.alpha.-factor) is synthesized as a prepro form of 165 amino acids comprising a 19 amino acids long signal- or prepeptide followed by a 64 amino acids long "leader" or propeptide, encompassing three N-linked glycosylation sites followed by (LysArg(Asp/Glu, Ala).sub.2-3 .alpha.-factor).sub.4 (Kurjan, J. and Herskowitz, I. Cell 30 (1982), 933-943). The signal-leader part of the preproMF.alpha.1 has been widely employed to obtain synthesis and secretion of heterologous proteins in S. cerivisiae.
Use of signal/leader peptides homologous to yeast is known from among others U.S. Pat. No. 4,546,082, European published patent applications Nos. 0116201A, 0123294A, 0123544A, 0163529A, and 0123289A and DK patent specifications Nos. 2484/84 and 3614/83.
In EP 0123289A utilization of the S. cerevisiae a -factor precursor is described whereas DK 2484/84 describes utilization of the Saccharomyces cerevisiae invertase signal peptide and DK 3614/83 utilization of the Saccharomyces cerevisiae PH05 signal peptide for secretion of foreign proteins.
U.S. Pat. No. 4,546,082, EP 0016201A, 0123294A, 0123544A, and 0163529A describe processes by which the .alpha.-factor signal-leader from Saccharomyces cerevisiae (MF.alpha.1 or MF.alpha.2) is utilized in the secretion process of expressed heterologous proteins in yeast. By fusing a DNA sequence encoding coding the S. cerevisiea MF.alpha.1 signal/leader sequence at the 5' end of the gene for the desired protein secretion and processing of the desired protein was demonstrated.
EP 206,783 discloses a system for the secretion of polypeptides from S. serevisiae whereby the .alpha.-factor leader sequence has been truncated to eliminate the four .alpha.-factor peptides present on the native leader sequence so as to leave the leader peptide itself fused to a heterologous polypeptide via the .alpha.-factor processing site LysArgGluAlaGluAla. This construction is indicated to lead to an efficient process of smaller peptides (less than 50 amino acids). For the secretion and processing of larger polypeptides, the native .alpha.-factor leader sequence has been truncated to leave one or two .alpha.-factor peptides between the leader peptide and the polypeptide.
A number of secreted proteins are routed so as to be exposed to a proteolytic processing system which can cleave the peptide bond at the carboxy end of two consecutive basic amino acids. This enzymatic activity is in S. cerevisiae encoded by the KEX 2 gene (Julius, D. A. et al., Cell 37 (1984b), 1075). Processing of the product by the KEX 2 gene product is needed for the secretion of active S, cerevisiae mating factor .alpha.1 (MF.alpha.1 or .alpha.-factor) but is not involved in the secretion of active S. cerevisiae mating factor a.
Secretion and correct processing of a polypeptide intended to be secreted is obtained in some cases when culturing a yeast organism which is transformed with a vector constructed as indicated in the references given above. In many cases, however, the level of secretion is very low or there is no secretion, or the proteolytic processing may be incorrect or incomplete. The present inventors currently believe this to be ascribable, to some extent, to an insufficient exposure of the processing site present between the C-terminal end of the leader peptide and the N-terminal end of the heterologous protein so as to render it inaccessible or, at least, less accessible to proteolytic cleavage.